A great deal of progress has been made in the past few years towards understanding a coherent mechanism of transcription elongation for E. coli RNA polymerase. The new insight came with the realization that the multi- domain polymerase performs catalysis and translocation along the template in units of single or multiple nucleotides, resulting from its ability to undergo monotonic or inchworm-like movement during elongation. The discontinuous movement confers upon the enzyme the ability to sense sequence-specific signals during transcription to bring about regulatory consequences, such as pausing and termination. In contrast, a mechanism of transcription initiation is not yet available, largely due to many unresolved issues associated with the promoter clearance phase. Promoter clearance phase spans RNA chain initiation and formation of phosphodiester bonds over the early position of the template to the point of sigma factor release from the enzyme-template complex. This phase is burdened with a high level of production of abortive transcripts, indicative of instability of the initial transcribing complexes. many determinants of promoter clearance have been elucidated that affect the ease of passage of the polymerase through this phase. The proposed research is aimed at understanding the effect of these determinants, with an emphasis on examining the role of initial transcribed sequence, template supercoiling, and the presence of GreA and GreB transcript cleavage factors. The specific aims of this proposal are three-fold: One, to identify features of the initial transcribed sequence that affect promoter clearance from the T5 N25 promoter. Two, to investigate the effect of template supercoiling and the role of transcript cleavage factors GreA and GreB, to probe the in vivo relevance of abortive initiation. Three, to determine the position of sigma factor release by photocrosslinking analysis. Together, these lines of investigation will lend clarification to many aspects of the promoter clearance process, and in turn, the mechanism of transcription initiation.